Surfactant Model

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Langmuir 1997, 13, 525-532

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Hydration and Structural Properties of Mixed Lipid/ Surfactant Model Membranes B. Ko¨nig,† U. Dietrich, and G. Klose* Department of Physics, University of Leipzig, Linne´ strasse 5, 04103 Leipzig, Germany Received June 10, 1996. In Final Form: October 22, 1996X Hydration properties of mixtures of a zwitterionic lipid, palmitoyloleoylphosphatidylcholine (POPC), and nonionic surfactants (oligo(oxyethylene) dodecyl ether, C12En with n ) 1-8) were studied over a wide range of surfactant/lipid molar ratios RA/L from 0.1 to 2 at T ) 25 °C. The adsorption of water by the POPC/C12En mixtures was measured by the isopiestic method at two different relative humidities (RH ) 86.5 and 97%). Deuterium NMR on 2H2O and 31P NMR on the phospholipid as well as X-ray diffraction were employed to characterize the phase state of the mixtures. For samples in the LR phase the area requirement of POPC and surfactant molecules and the thickness of the hydrophobic core of the bilayer were estimated from the repeat spacing and the known composition of the sample. The experimental results are compared to data reported previously for pure POPC and C12En systems under identical conditions. Small C12En concentrations (RA/L ) 0.1 and 0.2) in the membrane tighten the membrane packing. The area per molecule in the bilayer/water interface occupied by the lipid is reduced and that of the surfactant enlarged in the mixture compared to bilayers of the pure components under equal conditions. Further increase of the surfactant concentration causes a significant thinning of the hydrophobic core and a progressive increase of the area requirement of the amphiphilic molecules in the membrane/water interface. Finally, at high surfactant concentrations (RA/L ) 1 and 2) the area requirement of the amphiphilic constituents and the vertical extension of the polar interface region increase with growing ethylene oxide chain length n. The hydration of the lipid is reduced by the presence of C12En to a level comparable to the primary hydration shell. The first two or three oxyethylene groups next to the alkyl chain of the surfactant also show reduced hydration in the mixtures. The remaining EO groups have hydration characteristics very similar to the pure surfactant, with the exception of bilayers with RA/L ) 2 at RH ) 97%.

1. Introduction Investigations on phospholipid bilayers as model biological membranes have greatly increased our knowledge of fundamental membrane properties.1 The polar interface region of lipid membranes is a key factor for determining membrane features such as membrane integrity, interbilayer interactions and the ability of membranes to undergo fusion. The complexity of hydration of biologically relevant molecules continues to make this issue controversial.2 Theoretical models describing the hydration force between membranes have been suggested, emphasizing the role of structure and dynamics of the interface region from different perspectives.3,4 Nonionic lipid/surfactant mixed bilayer systems have been employed as models in studies on the hydration force5,6 and on membrane solubilization.7,8 In particular, modification of membrane physical properties may be accomplished easily with surfactants CmEn. Binary lipid/ CmEn mixtures can be varied through the surfactant by changing the size of the hydrophobic (m) or the hydrophilic (n) part of the molecule or by changing the lipid component. When studying the hydration properties of mixed membranes it is necessary to quantitate the amount of water * Corresponding author: e-mail, [email protected]. † Present address: Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institute of Health, Rockville, MD 20852. X Abstract published in Advance ACS Abstracts, December 15, 1996. (1) Cevc, G. In Phospholipid Handbook, Part II: Physical and Structural Properties; Marcel Dekker, Inc.: New York, 1993. (2) Israelachvili, J.; Wennerstro¨m, H. Nature 1996, 379, 219-225. (3) Israelachvili, J. N.; Wennerstro¨m, H. J. Phys. Chem. 1992, 96, 520-531. (4) Cevc, G. J. Chem. Soc., Faraday Trans. 1991, 87, 2733-2739. (5) Ko¨nig, B. PhD Thesis, University of Leipzig, 1993. (6) Ko¨nig, B.; Klose, G. Prog. Colloid Polym. Sci. 1993, 93, 279-280. (7) Thurmond, R. L.; Otten, D.; Brown, M. F.; Beyer, K. J. Phys. Chem. 1994, 98, 972-983. (8) Otten, D.; Lo¨bbecke, L.; Beyer, K. Biophys. J. 1995, 68, 584-597.

bound to each of the components of the membrane. A single spectroscopic technique is not capable of this measurement for a given mixed system. However, by systematically changing the length of the hydrophilic part of an oligomeric surfactant as well as the lipid/surfactant molar ratio, it should be possible to determine which fraction of the total amount of water adsorbed by the mixture can be assigned to lipid and surfactant, respectively. Mixtures of POPC and C12En with n ) 1 to 8 have been chosen for this investigation, since data on water sorption,9,10 phase behavior,11 structural features,8,9 and dynamical aspects12,13 of the pure components are available. Structural parameters of selected POPC/C12En systems in the lamellar liquid crystalline phase (LR) have been determined previously by neutron diffraction14 and fluorescence techniques.15 Amphiphiles without net electrical charge were used in order to avoid complications due to electrostatic head group interactions. Also, it was expected that the LR phase would prevail over a wide range of POPC/C12En composition.16,17 Mixtures of phospholipids and alkyl(oxyethylene) detergents are commonly associated during the nondestructive isolation and purification of membrane proteins by means of detergent solubilization of biological membranes (9) Klose, G.; Ko¨nig, B.; Paltauf, F. Chem. Phys. Lipids 1992, 61, 265-270. (10) Klose, G.; Eisenbla¨tter, S.; Galle, J.; Islamov, A.; Dietrich, U. Langmuir 1995, 11, 2889-2892. (11) Mitchell, D. J.; Tiddy, G. J. T.; Waring, L.; Bostock, T.; McDonald, M. P. J. Chem. Soc., Faraday Trans 1 1983, 79, 975-1000. (12) Rendall, K.; Tiddy, G. J. T. J. Chem. Soc., Faraday Trans 1, 1984, 80, 3339-3357. (13) Volke, F.; Eisenbla¨tter, S.; Galle, J.; Klose, G. Chem. Phys. Lipids 1994, 70, 121-131. (14) Klose, G.; Islamov, A.; Ko¨nig, B.; Cherezov, V. Langmuir 1996, 12, 409-415. (15) Lantzsch, G.; Binder, H.; Heerklotz, H.; Wendling, M.; Klose, G. Biophys. Chem. 1996, 58, 289-302. (16) Klose, G.; Eisenbla¨tter, S.; Ko¨nig, B. J. Colloid Interface Sci. 1995, 172, 438-446. (17) Funari, S. S.; Klose, G. Chem. Phys. Lipids 1995, 75, 145-154.

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and during reconstitution of proteins in model membranes.18,19 Even though the present study is focused on gaining insight in the complex interrelationships between structure, hydration, and phase behavior of lipid/surfactant mixtures on a very basic level, systematic investigations of such aggregates have a potential to improve the understanding of practical applications of lipid/surfactants mixtures. 2. Materials and Methods The phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was synthesized in the laboratory of Professor F. Paltauf at the Technical University in Graz, Austria. The purity of the lipid was checked by thin layer chromatography. No indication of lipid degradation was found. The ethylene oxide mono-n-dodecyl ethers C12H25(OCH2CH2)nOH (C12En) with n ) 1-8 were purchased from Nikko Chemicals (Tokyo, Japan) and used as received. The purity of the compounds was better than 99% based on gas chromatograms provided by the manufacturer. Prior to sample preparation lipid and surfactant were dehydrated by storing them in a desiccator in the presence of dry P2O5 for 24 h. The residual water content was less than one water per lipid or surfactant molecule, respectively, according to Karl Fischer coulometric titration. POPC/C12En mixtures with the following surfactant to lipid molar ratios (RA/L) were studied: 0.1, 0.2, 0.5, 1, and 2. Approximately 20 mg of POPC was mixed with the appropriate amount of C12En in ethanol. The solvent was evaporated under a stream of nitrogen, and the mixtures were dried further under medium vacuum conditions (3 × 10-2 Torr) for 6 h. In order to study hydration properties of POPC/ C12En mixtures, samples were stored under constant relative humidity (RH) conditions for 72 h in sealed vessels at 25 °C. The vessels contained a reservoir with a saturated solution of KCl (RH ) 86.5%) or K2SO4 (RH ) 97.0%) in 2H2O. Water exchange occurred exclusively via the vapor phase. Care was taken to ensure a temperature stability of better than (0.02 K in the vessels during the equilibration period. Further experimental details have been published previously.9,10 The amount of water adsorbed by the individual POPC/C12En samples was determined by weighing. After equilibration at RH ) 86.5% the sample tubes were temporarily sealed with Parafilm and NMR measurements were conducted. The samples were then subject to another equilibration period at RH ) 97%, followed by weighing and NMR studies. Finally, the hydrated POPC/C12En mixtures were transferred into thin-walled glass capillaries (1 mm outer diameter) which were sealed after filling and X-ray diffraction studies were performed. The evaluation of the phase state of hydrated POPC/C12En mixtures was based on the line shape of the 31P NMR signal of the lipid and on the 2H NMR spectra of 2H2O. A detailed discussion of NMR line shape analysis for determination of the phase state of lipids20,21 and in particular of POPC/C12En mixtures16,17 can be found in the literature. The 31P NMR spectra were recorded at 121.4 MHz under continuous broadband proton decoupling and the 2H NMR spectra at 46.07 MHz, both on a Bruker MSL300 spectrometer with a field strength of 7.04 T. X-ray diffraction was applied to determine the lamellar repeat spacing d of the samples equilibrated at RH ) 97%. The small angle diffraction pattern was also used to reconfirm the presence of lamellar and/or hexagonal phase structures as seen by NMR methods when applicable.22 The sample capillaries were mounted into an adjustable temperature brass sample holder at 25 °C in front of a pinhole collimator. The nickel-filtered Cu KR line (λ ) 0.1542 nm) was used. Diffraction patterns were recorded with (18) Carvell, M.; Hall, D. G.; Lyle, I. G.; Tiddy, G. J. T. Faraday Discuss. Chem. Soc. 1986, 81, 223-237. (19) Moller, J. V.; LeMaire, M.; Andersen, J. P. In Progress in ProteinLipid-Interactions; Watts, A., DePont, J. J. H. H. M., Eds.; Elsevier/ North Holland: Amsterdam, 1986; Vol. 1, pp 147-196. (20) Cullis, P. R.; deKruijff, B. Biochim. Biophys. Acta 1979, 559, 399-420. (21) Chan, S. I.; Bocian, D. F.; Petersen, N. O. In Membrane Spectroscopy; Grell, E., Ed.; Springer-Verlag: Berlin, Heidelberg, 1981; pp 1-50. (22) Luzzati, V. In Biological Membranes; Chapman, D., Ed.; Academic Press: London and New York, 1968; Vol. 1, pp 71-124.

Ko¨ nig et al. a linear position sensitive detector (Braun, Germany). The distance between sample and detector was about 18 cm. A helium gas filled cylinder with Mylar windows was placed between sample and detector to reduce “air-scattering”. The precision of the repeat distances determined was better than (0.05 nm. The lamellar repeat spacing d can be used to calculate additional geometrical features of the membrane stack under consideration by using an approach originally proposed by Luzzati.22 Note that the method is applicable only if all the components of the membrane are arranged in a uniform multilamellar fashion, i.e. lateral phase separation or clusters of free water must not be present.23 The mean lateral surface area AL+A occupied by one POPC plus RA/L surfactant molecules at the membrane/water interface is

AL+A ) 2(υL + RA/LυA + RW/LυW)/d

(1)

where υi ) Miνi/NA are molecular volumes of lipid (i ) L), surfactant (i ) A), and water (i ) W), respectively. RA/L is the molar surfactant to lipid ratio and RW/L that of the total amount of water sorbed by one lipid plus RA/L surfactant molecules and lipid. Mi and νi are the molecular weight and the partial specific volume of the components, respectively, and NA is Avogadro’s number. The molecular weights of the components used are as follows (in g/mol): 760 (POPC), 20 (2H2O), 230 (C12E1), 274 (C12E2), 318 (C12E3), 362 (C12E4), 406 (C12E5), 450 (C12E6), 494 (C12E7), and 538 (C12E8). νL and νA were taken to be 1 cm3/g, and for 2H2O a value of νW ) 0.9 cm3/g was used. Further, the thickness of the hydrophobic membrane core dhc can be estimated according to

dhc ) dυhc/(υL + RA/LυA + RW/LυW)

(2)

where υhc is the molecular volume of the hydrocarbon chains of one POPC molecule (M ) 448 g/mol) plus RA/L times the molecular volume of a surfactant dodecyl chain (M ) 169 g/mol). The partial specific volume of that hydrocarbon was taken to be 1.17 cm3/ g.24,25

3. Results Phase Behavior. Most of the samples studied were found to be in the lamellar liquid-crystalline phase (LR) with no indication of additional phases. The 2H NMR spectra of these samples showed a single Pake doublet, characteristic for anisotropic mesophases. No indication for isotropic water was found in the spectra. One might anticipate qualitatively distinctive water binding sites in POPC/C12En mixtures. However, a given water molecule must undergo an exchange between these sites which is fast on the NMR time scale (10-3-10-4 s for these measurements) in order to account for the spectra obtained. The 2H NMR spectra confirm the absence of a free water subphase in the samples. Also, the presence of just one Pake doublet per spectrum strongly indicates the absence of lateral segregation of lipid and surfactant on a macroscopic scale. To be consistent with the spectra, the time-averaged quadrupolar splitting of the 2H spins of the water would have to be the same for the separate domains. This is a rather unlikely case for all the samples over the entire RA/L range studied. The 31P NMR spectra of the same samples gave rise to lineshapes also characteristic of randomly oriented bilayers in the LR phase.20,21 The observed chemical shift anisotropy was close to the value of ∆σ ) -46 ( 2 ppm reported by Bechinger and Seelig26 for POPC with RW/L between 4 and 22 at 23 °C. The small angle X-ray measurements gave typical lamellar patterns for these samples; i.e., the spacings derived from the diffractogram were related to the fundamental repeat (23) Klose, G.; Ko¨nig, B.; Meyer, H. W.; Schulze, G.; Degovics, G. Chem. Phys. Lipids 1988, 47, 225-234. (24) Nagle, J. F.; Wilkinson, D. A. Biophys. J. 1978, 23, 159-175. (25) Rand, R. P.; Parsegian, V. A. Biochim. Biophys. Acta 1989, 988, 351-376. (26) Bechinger, B.; Seelig, J. Chem. Phys. Lipids 1991, 58, 1-5.

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Langmuir, Vol. 13, No. 3, 1997 527 Table 1. Differential Water Uptake per EO Segment (aW*) and Number of Water Molecules Adsorbed by one POPC in a Hypothetical Mixture with RA/L Dodecanol Molecules (bW) Obtained by Linear Regression of Water Sorption Data of POPC/C12En Mixtures at T ) 25 °C and RH ) 97% According to Equation 3 RH 97%

86.5%

a

Figure 1. Number of water molecules WL+A adsorbed per POPC plus RA/L C12En molecules at 25 °C under relative humidity conditions of 86.5% (below) and 97% (above), respectively, as a function of the number of the ethylene oxide units n of the surfactant. Experimental values for molar surfactant/lipid ratios RA/L of 0.1 (1), 0.2 (9), 0.5 ([), 1 (b), and 2 (2) are shown. Filled symbols are used for samples in the LR phase, while open symbols indicate the presence of both lamellar and hexagonal phases. The lines included are the result of linear regression. The experimental uncertainty of WL+A is (1.5 water molecules.

distance, d, of the stack by factors of 1/2, 1/3, 1/4, .... Wide angle X-ray studies have been performed previously on some selected POPC/C12En samples at 25 °C, and a very broad reflection centered at 0.45 ( 0.01 nm was always observed, which is characteristic for the LR phase.5 For samples with very high C12En content and short ethylene oxide chains a mixture of two phases was observed. Both the 2H and 31P NMR spectra were consistent with the coexistence of lamellar and hexagonal phases.16,17 The small angle X-ray patterns did show at least one strong reflex in addition to the lamellar type series of peaks. The samples showing the two-phase characteristics are marked in Figure 1 by using open symbols. We could not use the Luzzati approach for a detailed geometrical description of the mesophases present in the two-phase region, since the composition of each of these phases is not known. Hydration Properties. The amount of water adsorbed by one POPC plus RA/L surfactant molecules denoted by WL+A analogously to AL+A is presented as a function of the number of ethylene oxide subunits of the surfactant for five different surfactant/lipid ratios at RH values of 86.5% and 97%, respectively, in Figure 1. For some of the RA/L values studied, WL+A did change in a fairly linear fashion as a function of n, either over the entire range of 1 e n e 8 or for a limited region only. In these cases a linear regression of the data points was performed.

WL+A ) aW n + bW

(3)

The parameter bW can formally be considered as a hypothetical measure of the amount of water adsorbed by one lipid molecule when mixed with RA/L dodecanol molecules at the given relative humidity. The slope of the regression line aW allows one to access the differential water uptake per ethylene oxide segment aW* ) aW/RA/L.

RA/L

aW*

bW

n range

2 1 0.5 0.2 0.1 pure lipid pure surfactant 2 1 0.5 pure lipid pure surfactant

1.3 ( 0.2 2.2 ( 0.3 2.3 ( 0.4 2.3 ( 0.3 4.9 ( 1.9

10.9 ( 1.8 7.7 ( 1.4 7.8 ( 1.0 7.8 ( 0.3 5.9 ( 0.9 13.5a

2-8 2-8 2-8 2-8 2-8

6.2 ( 0.8 6.9 ( 0.8 6.2 ( 1.0 7.5a

1-8 1-8 1-8

2.1b 0.7 ( 0.1 0.7 ( 0.2 0.8 ( 0.4 0.7b

Values from ref 9. b Values from ref 10.

The water sorption of POPC/C12En mixtures at RH ) 86.5% seems to be a linear function of n over the entire n-range studied for RA/L ) 2, 1, and 0.5, even though in the latter case the scatter of the data points is rather big (Figure 1, below). The aW* values determined (cf. Table 1) are in good agreement with (for RA/L ) 2 and 1) or at least close to (for RA/L ) 0.5) the differential water uptake of 0.7 water molecules per additional EO-segment determined previously for pure C12En samples under identical conditions.10 The values obtained for bW (cf. Table 1) are marginally lower than the corresponding WL+A ) 7.5 ( 1, which was measured for pure POPC.9 The water sorption data for RH ) 86.5% at the lower surfactant concentrations scatter appreciably and systematic changes of WL+A as a function of n, if any, are within the range of experimental error of the measurements. For this reason, no linear regression of the data was performed. However, our data do indicate that the water sorption per POPC plus RA/L C12En molecules at RA/L ) 0.1 and 0.2 is slightly less than the hydration of pure POPC under identical conditions. The amount of water taken up by the POPC/C12En mixtures at RH ) 97% did not change much for EOn chain length between 1 and 3 for given RA/L. There is clearly a linear tendency of WL+A upon n in the n-range from 2 or 3 to 8 for all surfactant/lipid ratios studied (Figure 1, above). Linear regression was performed using the data with 2 e n e 8; however exclusion of the data points for n ) 2 insignificantly changed the fit parameters given in Table 1. The differential water uptake aW* for the mixtures with RA/L values 0.2, 0.5, and 1 is very close to the value 2.1 determined previously for pure C12En samples under identical conditions.10 A higher value was found for RA/L ) 0.1, and even more interesting, a lower differential water uptake was measured for RA/L ) 2. The hypothetical water uptake bW for n ) 0 was considerably lower than the water sorption of pure POPC under identical conditions (RW/L ) 13.5 ( 1)9 for all RA/L values studied (Table 1). Structural Parameters. The repeat spacing d determined by X-ray diffraction for POPC/C12En mixtures hydrated at RH ) 97% and T ) 25 °C is shown as a function of the length n of the EO chain in the upper part of Figure 2. Only data obtained for samples exhibiting a single lamellar phase are included. For given RA/L the d values change with n in a rather linear fashion. The straight lines shown in Figure 2 were obtained by linear regression of the data points. At low surfactant concentrations (RA/L ) 0.1 and 0.2) d decreases slightly with the length n of the EO chain and almost all mixtures show a larger spacing than pure POPC under identical conditions (d ) 5.18 nm).9

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Ko¨ nig et al.

Figure 3. Area requirement per POPC plus RA/L C12En molecules AL+A at the membrane/water interface vs number of ethylene oxide units n of the surfactant. Data shown are from samples in the LR phase only, which were hydrated at T ) 25 °C and RH ) 97%. Molar surfactant/lipid ratios are 0.1 (1), 0.2 (9), 0.5 ([), 1 (b), and 2 (2). Lines were obtained by linear regression.

Figure 2. Repeat spacing d (above) and thickness of the hydrocarbon core dhc (below) of POPC/C12En membranes hydrated at T ) 25 °C and RH ) 97% vs number of ethylene oxide units n of the surfactant. Molar surfactant/lipid ratios are 0.1 (1), 0.2 (9), 0.5 ([), 1 (b), and 2 (2). Data from samples in the LR phase only are included. Lines were obtained by linear regression. The experimental uncertainty is (0.05 nm.

At RA/L ) 0.5 the negative slope of d vs n is steeper and for the longer EO chains d plunges below the value obtained for pure POPC. The d values for RA/L ) 1 and 2 are reduced even further and they show the tendency to increase with growing n in contrast to the behavior of the mixtures with lower RA/L. The thickness of the hydrophobic membrane core dhc calculated from the d values according to eq 2 is shown in the lower part of Figure 2. The dhc values decrease monotonically with n for all the surfactant/lipid ratios studied. The dependence of dhc on n seems to be fairly linear over the entire n-range investigated. The values of the slope of the straight lines in Figure 2 (below) are quite similar. Only mixtures with RA/L ) 0.5 exhibit different behavior. Perhaps there is a break in the dependence of dhc on n between n ) 3 and 4. Separate linear regression of the data points in the intervals 1 e n e 3 and 4 e n e 8, respectively, gives values for the slope close to those observed for the other mixtures. A value of dhc ) 2.72 nm was calculated for pure POPC based on previously published results.9 The dhc data shown in Figure 2 (below) for POPC/C12En mixtures for RA/L ) 1 and 2 are generally smaller, while for RA/L ) 0.1 and 0.2 they are generally greater than the value for pure POPC. For RA/L ) 0.5 there is a crossover in this relationship between n ) 3 and 4. The mean lateral surface area AL+A occupied by one POPC plus RA/L surfactant molecules for samples hydrated at RH ) 97% was calculated according to eq 1 and is shown in Figure 3. AL+A vs n increases in a fairly linear way for given RA/L over the entire range studied. Regression of the data was performed according to eq 4.

AL+A ) aAn + bA

(4)

The parameter bA can formally be considered as a hypothetical measure of the area requirement of one lipid molecule plus RA/L dodecanol molecules. The slope aA

Figure 4. Area requirement per POPC plus RA/L C12En molecules AL+A at the membrane/water interface vs surfactant/ lipid ratio RA/L for n ) 2 (9), 4 (b), 6 (2), and 8 (1).

allows one to determine the differential area increase upon addition of one ethylene oxide segment aA* ) aA/RA/L. All the AL+A data calculated for RA/L ) 0.1 are smaller than the surface area per lipid molecule of 0.64 ( 0.01 nm2 determined for POPC also hydrated at 25 °C and RH ) 97%.9 For higher surfactant concentrations (RA/L from 0.5 to 2) the AL+A data of the mixtures are larger than the POPC value. The differential area increase aA* for mixtures with RA/L e 0.5 is larger than the area increase of 0.037 nm2 per EO segment observed for pure C12En under identical conditions.10 One can obtain estimates of the partial surface requirements of the POPC and of the surfactants in the mixed membranes by analysis of the dependence of the mean surface area AL+A on the surfactant concentration RA/L. Obviously, the AL+A vs RA/L data in the ranges 0.1 e RA/L e 0.5 and 0.5 e RA/L e 2 can be quite well approximated by linear equation (Figure 4)

AL+A ) cARA/L + dA

(5)

Our data indicate that the partial surface requirements of lipid and surfactant in the mixtures are constant over an extended concentration range and behave in an additive fashion. Therefore, the parameter dA gives the partial surface requirement of the lipid and the slope cA that of the surfactant. The results of the regression of the data according to eq 5 together with literature values available for the surface requirements of the pure surfactant under the same conditions are collected in Table 3.

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Table 2. Differential Area Increase per EO Segment (aA*) and Area per POPC Plus RA/L Hypothetical Dodecanol Molecules (bA) Obtained by Linear Regression of Data Shown in Figure 3 According to Equation 4 RA/L

aA*/nm2

bA/nm2

n range

2 1 0.5 0.2 0.1 pure lipid

0.034 ( 0.005 0.041 ( 0.004 0.065 ( 0.004 0.065 ( 0.008 0.093 ( 0.01

1.11 ( 0.06 0.81 ( 0.03 0.62 ( 0.01 0.59 ( 0.01 0.55 ( 0.01 0.64a

4-8 4-8 1-8 1-8 1-8

a

Value from ref 5.

The analysis at low (RA/L e 0.5) surfactant concentration yields a mean surface area of 0.55 nm2 with a mean deviation of (0.01 nm2 for POPC in the mixtures which is considerably smaller than the value for pure POPC (0.64 nm2), and the surface areas of the surfactants in the mixtures have a tendency to be larger than the values for pure surfactants. Alternatively one can use the values of the hypothetical area requirement of one POPC molecule plus RA/L dodecanol molecules, bA (Table 2), to calculate the partial area requirement per lipid in the mixtures. Assuming a partial area requirement per dodecanol molecule of 0.23 nm2, this yields, on average, a value of 0.53 ( 0.02 nm2, which matches the 0.55 ( 0.01 nm2 derived from eq 5 at RA/L e 0.5 within experimental error. At high surfactant concentration (0.5 e RA/L e 2) the values of the partial area requirement per POPC molecule are larger than 0.55 nm2 (cf. parameter dA in Table 3) and they increase with the length of the EO chain. For n ) 8 the parameter dA has reached a value close to the area requirement of POPC in the pure lipid bilayer (0.64 nm2). The values of the partial surface requirement of the surfactant, cA , are smaller than those obtained at low surfactant concentration (RA/L e 0.5) but close to the area per C12En molecule determined for the pure surfactant bilayer under identical conditions.10 4. Discussion First we would like to address the issue of whether the hydration characteristics of the pure components POPC and C12En change upon forming mixed aggregates. In other words, does the water sorption of POPC and C12En behave in an additive fashion in POPC/C12En mixtures under identical sorption conditions? The sorption data obtained at RH ) 86.5% show only minor deviations from additiv uptake of water for mixtures with RA/L ) 2, 1, and perhaps 0.5. The similar water uptake per EO unit in pure C12En systems, which were studied previously,10 and in POPC/C12En mixtures indicates the absence of any significant changes in the hydration properties of the EO groups due to the anchoring of the hydrocarbon chain of the surfactant in the hydrophobic core of the mixed aggregate rather than forming an aggregate with other surfactant molecules only. There may be two exceptions to this general trend. First, increasing the number of EO units from 1 to 2 does not seem to increase the water sorption (Figure 1, lower part). This observation could be explained by a location for the first one or two EO groups beyond the glycerol moiety of the lipid toward the center of the bilayer, presumably in a fairly dehydrated state. A recent neutron diffraction study on POPC/C12En mixtures with deuterium-labeled EO groups has indeed suggested that one oxyethylene group might penetrate into the hydrocarbon region of the membrane even beyond the fatty acid ester group of the lipid.14 Also, the intrusion of polar EO moieties into the hydrocarbon core of surfactant

micelles was observed under certain circumstances.27 Second, a linear relationship between the water sorption vs n for given RA/L (Figure 1, lower part) intercepts the WL+A axis at bW (Table 1). Values obtained for bW in this fashion are slightly smaller than RW/L for pure POPC at RH ) 86.5%. This may be caused by partial replacement of water molecules in the hydration shell of the lipid headgroup with EO moieties. Ethylene glycol has been used as a polar solvent instead of water in lipid systems in the past.28,29 Also two-dimensional NMR studies on POPC/C12E4 mixtures suggested intimate contact between the lipid headgroup and EO segments.30 The results obtained at RH ) 86.5% with RA/L ) 0.1 and 0.2 are not consistent with an additive sorption behavior of the two components. The amount of water adsorbed by these mixtures was generally lower than the 7.5 molecules of water reported for pure POPC under identical conditions. This behavior cannot be accounted for by simple replacement of hydration water of the lipid with EO groups due to the low surfactant concentration. It is more likely caused by a slight change in the hydration characteristic of the lipid triggered by the small fraction of surfactant present in the mixture. Moreover, an evaluation of the differential water uptake per EO segment is outside the scope of the sensitivity of the sorption measurements for these very low surfactant concentrations. Some features of the sorption behavior discussed for RH ) 86.5% are encountered again at RH ) 97% (upper part of Figure 1). For given RA/L the amount of water adsorbed by POPC/C12En mixtures is nearly constant for EO chain length up to n ) 3, which might be due to a location of the first few EO segments below the polar lipid/ water interface in the hydrophobic region. The differential water uptake of the remaining EO moieties for mixtures with RA/L ) 1, 0.5, and 0.2 closely resembles the hydration of EO segments in pure C12En systems.10 An interpretation of the differential water uptake per EO segment for RA/L ) 0.1 is not possible due to the large error of aW* at low C12En concentration. The straight lines obtained by linear regression of the WL+A vs n data at RH ) 97% intercept the WL+A axis at values bW (Table 1) which are significantly lower than the water sorption of pure POPC at RH ) 97%. Thus, the water binding capacity of the lipid is sharply reduced by the presence of surfactant in the membrane. At the higher C12En concentrations the underlying mechanism might be a partial replacement of water molecules by EO segments. In any event, the sorption data obtained for POPC/C12En mixtures cannot be accounted for by simple additivity of the sorption of the two components. Our data indicate instead a partial dehydration of the lipid due to the presence of the surfactant. A zwitterionic phosphatidylcholine lipid headgroup can strongly bind water molecules, mainly by hydrogen bonding. The differential free energy gain resulting from binding a water molecule to a lipid headgroup decreases sharply with the number of water molecules already associated with the lipid.31 The gain in hydration free energy arises mainly from the water molecules directly bound, which are frequently referred to as primary hydration shell. One should expect this primary hydration layer to consist of six or seven water molecules for a POPC (27) Elworthy, P. H.; Patel, M. S. J. Pharm. Pharmacol. 1984, 36, 565-568. (28) McDaniel, R. V.; McIntosh, T. J.; Simon, S. A. Biochim. Biophys. Acta 1983, 731, 97-108. (29) Persson, P. K. T.; Bergenstahl, B. A. Biophys. J. 1985, 47, 743746. (30) Volke, F.; Pampel, A. Biophys. J. 1995, 68, 1960-1965. (31) Cevc, G.; Marsh, D. In Phospholipid Bilayers: Physical Principles and Models; Cevc, G., Marsh, D., Eds.; John Wiley & Sons, Inc.: New York, 1987; Chapter 3.

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Table 3. Partial Area Requirement of POPC (dA) and C12En (cA), Respectively, in POPC/C12En Mixtures at T ) 25 °C and RH ) 97% As Derived from Figure 4 by Linear Regression According to Equation 5a RA/L range 0.1-0.5

0.5-2.0

n

dA/nm2

cA/nm2

dA/nm2

cA/nm2

AAb/nm2

1 2 3 4 5 6 7 8 mean value

0.542 ( 0.021 0.531 ( 0.012 0.553 ( 0.009 0.545 ( 0.014 0.562 ( 0.01 0.54 ( 0.002 0.554 ( 0.003 0.557 ( 0.014 0.55 ( 0.01c

0.249 ( 0.066 0.295 ( 0.037 0.309 ( 0.029 0.466 ( 0.045 0.452 ( 0.032 0.583 ( 0.008 0.577 ( 0.01 0.653 ( 0.044

0.569 ( 0.001 0.576 ( 0.003 0.612 ( 0.008 0.586 ( 0.007 0.632 ( 0.001

0.412 ( 0.001 0.423 ( 0.002 0.447 ( 0.006 0.52 ( 0.005 0.499 ( 0.001

0.426 0.454 0.48 0.542

a Area requirement A of C E in pure surfactant bilayers10 is included for comparison. b Values from ref 10. c Pure lipid under the same A 12 n conditions AL ) 0.64 nm2 (cf. ref 5).

molecule in the LR phase based on simple geometric considerations. Consequently, the water sorption of pure POPC at RH ) 86.5% is roughly equivalent to the completion of the primary hydration shell. The rather pronounced distortion of the molecular packing of the lipid molecules induced by incorporation of up to 2-fold excess of surfactant molecules into the matrix only marginally decreased the hydration of the lipid at RH ) 86.5%. On the other hand, at RH ) 97% the hydration of the pure lipid (RW/L ) 13.5) clearly exceeds the primary hydration shell. The additional water is much more weakly bound. The data obtained for the mixed systems at RH ) 97% are consistent with the primary hydration shell of the lipid still present, but the weakly bound water has been replaced by hydrated EO segments. Thus, the weakly bound water does not appear to be a major barrier in the approach of polar lipid and surfactant moieties. The influence of the lipid on the hydration of the surfactant molecules seems to be restricted to the partial dehydration of the first few EO groups presumably due to incorporation in the nonpolar region. The features of lipid/surfactant hydration presented here fit well in the general picture of the role of hydration in colloidal systems published recently by Israelachvili and Wennerstro¨m.2 The sorption behavior for RA/L ) 2 at RH ) 97% is rather different from the cases discussed so far. The differential water uptake per EO group is reduced by nearly a factor of 2 in comparison with pure surfactant systems. Obviously the presence of the lipid component creates additional forces, which counteract the hydration of the EO moieties. For instance, the van der Waals attraction between adjacent bilayers may be higher than in pure surfactant systems. The X-ray data obtained at RH ) 97% add a structural dimension to the hydration behavior discussed above. Progressive dehydration of phosphatidylcholine lipids in the LR phase is known to increase the thickness of the hydrocarbon core and to decrease the lateral area requirement per lipid molecule in the bilayer.32 Both trends are found in POPC/C12En mixtures at RA/L ) 0.1 and 0.2 and might at least partially be accounted for by the reduction in the hydration of the lipid. The partial area requirement of the lipid in the mixtures (parameter dA in Table 3) at low surfactant concentration (0.1 e RA/L e 0.5) is independent of the EO chain length n within experimental error and amounts, on average, to 0.55 ( 0.01 nm.2 This corresponds to a POPC molecule which is slightly dehydrated beyond the level observed for pure POPC membranes at T ) 25 °C and RH ) 97%. In addition, there is probably a change of the mean confor(32) Janiak, M. J.; Small, D. M.; Shipley, G. G. J. Biol. Chem. 1979, 254, 6068-6078.

mation of the lipid molecules due to the presence of the surfactant which is reflected by the larger repeat spacings of the mixtures at RA/L ) 0.1 and 0.2 as compared to pure POPC (d ) 5.18 nm). The structural parameters observed might be caused by a reduced mobility of the acyl chains of the lipid, which would result in an increased average length and a lower area requirement per hydrocarbon chain. The partial area requirement of the surfactant (parameter cA in Table 3) at 0.1 e RA/L e 0.5 increases with growing n, and in cases where the pure surfactant also forms a liquid crystalline bilayer under these conditions its area requirement in the mixtures is slightly higher (Table 3). This might indicate a reduced lateral pressure experienced by the EO moieties in the mixtures and should result in a more disordered, more random coil-like structure of the EO chain. This hypothesis is also in agreement with the higher values of the differential area increase per EO-group (parameter aA* in Table 2) observed for the mixed systems as compared to 0.037 nm2 for pure C12En bilayer.10 Our results for the mixtures at low surfactant concentrations indicate the hydrated EO groups to be accommodated in the polar headgroup region of the lipid rather causing an additional thickening of this region since both d and dhc tend to decrease with growing n (Figure 2). At high surfactant concentration dhc is significantly smaller than the value of 2.72 nm determined for pure POPC. This is partially due to the mismatch of the length of the hydrocarbon chains of POPC and C12En which induces a considerable increase in disorder in the hydrocarbon core of the bilayer. The lateral steric repulsion of hydrated EO chains is expected to increase the surface requirement AL+A with both n and RA/L as well as to decrease dhc in order to maintain a constant mass density in the hydrocarbon region. In contrast to the membrane stacks with low C12En concentration, the repeat spacing d increases with the length of the EO chains for RA/L ) 1 and 2. Taken together with the decrease in dhc this indicates a progressive vertical extension of the hydrated polar region of the mixed bilayers with increasing n as a result of the incorporation of surfactant at high concentration. Consequently the enlargement of the combined area AL+A with increasing length of the EO chain in these cases is due both to a lateral expansion of the detergent molecules and to changes in the average conformation of the lipid triggered by the presence of the surfactant. The latter is reflected by the increased partial area requirement of POPC (parameter dA in Table 3) at high surfactant concentration. The proximity of the differential area increase per EO group at RA/L ) 1 and 2 (aA* in Table 2) to the 0.037 nm2 for pure surfactant systems10 is consistent with a structure of the EO chains in mixed bilayers similar to that present in pure surfactant bilayers.

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Properties of Lipid/Surfactant Membranes

Binary phase diagrams for a variety of C12En/water mixtures can be found in the literature.11,33,34 The LR phase is predominant at room temperature for systems with n ) 3 and 4 and also exists for n ) 5 to 8, however as a declining fraction. At n ) 6 a hexagonal phase HI kicks in the share of which increases with n. The tendency to prefer curved structures upon enlarging the polar part of the surfactant is predicted by the molecular packing concept introduced by Israelachvili et al.35,36,37 Under low hydration condition (RH ) 86.5%) the pure surfactants form either a micellar solution phase (n ) 6 to 8) or a surfactant liquid phase (n ) 2 to 5) while stronger hydration (RH ) 97%) brings about the transition in the LR phase (n ) 2 to 7) or in the HI phase (n ) 8).10,11 POPC and water at 25 °C form liquid crystalline bilayer structures over nearly the entire concentration range either as a homogeneous LR phase at reduced water content or as an equilibrium state with excess water.9 This behavior is in accordance with the nearly cylindrical molecular shape of POPC in terms of the packing concept. The majority of POPC/C12En mixtures studied here adopt the LR phase preferred by the lipid. However, depending on the hydrocarbon chain characteristics, a phosphatidylcholine monolayer can have a certain internal tendency to curl. This is frequently described in the frame of the intrinsic curvature concept of Gruner and co-workers.38,39,40 In bulk phosphatidylcholine systems formation of nonlamellar phases is usually not observed due to the high degree of hydrocarbon packing strain which would result otherwise. This obstacle can be overcome by adding alkane,41,42 which easily partitions into the hydrocarbon region of the lipid phase, thereby preventing the packing strain. The suggested ability of the first few EO segments to insert into a nonpolar environment14,27,30 may enable C12En molecules with low n to promote the formation of the liquid crystalline HII phase in a very similar fashion. Fairly high surfactant concentrations are required to trigger the formation of HII structures (Figure 1). Also, the hexagonal phase is only found at low water content, as shown previously for two POPC/C12En mixtures.16,17 This was explained with an increase of the molecular asymmetry of the partially dehydrated lipid, which should facilitate the transition into a nonlamellar phase.16 C12En with n ) 5 to 8 is known to cause solubilization of POPC vesicles after a saturating fraction of surfactant in the mixed bilayer is reached and the corresponding critical surfactant concentrations have been determined in excess water at 25 °C.43,44 For n ) 6, 7, and 8 they are below the maximum surfactant concentration covered by the present study. However, the corresponding NMR spectra did not show any indication of an isotropic (micellar) phase at high RA/L. The implication is that (33) Conroy, J. P.; Hall, C.; Leng, C. A.; Rendall, K.; Tiddy, G. J. T.; Walsh, J.; Lindblom, G. Prog. Colloid Polym. Sci. 1990, 82, 253-262. (34) Clunie, J. S.; Goodman, J. F.; Symons, P. C. Trans. Faraday Soc. 1969, 65, 287-296. (35) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525-1568. (36) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Biochim. Biophys. Acta 1977, 470, 185-201. (37) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. Rev. Biophys. 1980, 13, 121-200. (38) Kirk, G. L.; Gruner, S. M.; Stein, D. L. Biochemistry 1984, 23, 1093-1102. (39) Gruner, S. M. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 36653669. (40) Kirk, G. L.; Gruner, S. M. J. Phys. (Paris) 1985, 46, 761-769. (41) Sjo¨lund, M.; Lindblom, G.; Rilfors, L.; Arvidson, G. Biophys. J. 1987, 52, 145-153. (42) Sjo¨lund, M.; Rilfors, L.; Lindblom, G. Biochemistry 1989, 28, 1323-1329. (43) Heerklotz, H.; Binder, H.; Lantzsch, G.; Klose, G. Biochim. Biophys. Acta 1994, 1196, 114-122. (44) Heerklotz, H. PhD Thesis, University of Leipzig, 1996.

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Figure 5. Deuterium quadrupolar splitting ∆νQ of 2H2O measured on mixtures of POPC/C12En hydrated at RH ) 97% (above) and RH ) 86.5% (below), respectively, as a function of the number of ethylene oxide units n of the surfactant at T ) 25 °C. Data from samples in the LR phase are shown only. Molar surfactant/lipid ratios are 0.1 (1), 0.2 (9), 0.5 ([), 1 (b), and 2 (2). The experimental uncertainty is (50 Hz.

reduced hydration decreases or perhaps even disables the solubilization potential of the surfactants studied for POPC. The deuterium quadrupolar splitting of 2H2O in membranes is directly proportional to an order parameter S which describes the motionally averaged orientation of the O-2H bonds with respect to the bilayer normal.45 Fast exchange of 2H2O molecules between qualitatively distinct binding sites on the NMR time scale of the experiment results in spectra showing just one Pake doublet.13,46 Although a dynamically averaged quantity like ∆νQ cannot provide a molecularly resolved description of the water binding polar region of the membrane, comparison of the data obtained in the present study for lamellar POPC/ C12En mixtures (Figure 5) with ∆νQ values reported in the literature for pure POPC13 and C12En12 in the LR phase as a function of water concentration is useful. The ∆νQ literature data can be linked with RH values by using the known water sorption characteristics of the amphiphiles.9,10 The quadrupolar splitting for POPC/2H2O narrows with increasing water content and values of =830 Hz and =450 Hz were found at RH ) 86.5 and 97%, respectively.13 The parameter ∆νQ decreases in an approximately linear manner from =2700 Hz (n ) 3) to =1000 Hz (n ) 6) in lamellar C12En/2H2O systems at RH ) 97% and T ) 25 °C.12 The steep decline of ∆νQ with growing n is caused by a large decrease of the order parameters for the EO groups on increasing distance from the dodecyl chain of the surfactant.12 Qualitatively the same behavior of ∆νQ vs n was found for POPC/C12En systems at RA/L ) 1 and 2 (Figure 5). This indicates motional characteristics of hydrated EO segments, which resemble those present in pure surfactant bilayers. The absolute values of ∆νQ are lower for mixtures than for C12En lamellae most likely due to fast exchange of water between binding sites on lipid and surfactant. As expected the reduction of ∆νQ is more pronounced for the lower surfactant concentration (45) Seelig, J.; Seelig, A. Q. Rev. Biophys. 1980, 13, 19-61. (46) Finer, E. G.; Darke, A. Chem. Phys. Lipids 1974, 12, 1-16.

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RA/L ) 1. The series with RA/L ) 0.1, 0.2, and 0.5 behave in a different manner. The ∆νQ data for the mixtures are again higher than the values determined for pure POPC under identical conditions, but there is very little influence of RA/L on the data and ∆νQ decreases only slightly with growing n. The rather big jump in ∆νQ after addition of small amounts of surfactant could be due to changes in the average conformation of the lipid headgroup or reduction in headgroup hydration. Both processes are known to be correlated26,47 and either one of them is expected to be reflected in changes of the experimental parameter ∆νQ.13 The rather small variation in ∆νQ with RA/L also supports the hypothesis that C12En at low concentration influences the bilayer hydration mainly by triggering a change in conformation and/or hydration of the lipid component. In summary, our water sorption, X-ray, and NMR data suggest a partial dehydration of the lipid headgroup as a result of addition of even small amounts of surfactant at RH ) 86.5% and more pronounced at RH ) 97%. Dehydration may be caused by hydrogen bond formation of the terminal OH of the EO chain with the phosphate moiety of POPC or more likely by replacement of weakly bound water in excess of the primary hydration shell of the lipid. Also an insertion of a few EO groups into the nonpolar membrane region might contribute to a reduced water binding by the surfactant. A tightening of the lipid packing at small C12En concentrations in the membrane, which can be concluded from the X-ray data, might explain the reduced water permeation into these bilayers observed by fluorescence spectroscopy.15 Further increase of the surfactant concentration resulted in a significant decrease in membrane thickness and in a progressively larger lateral area requirement per lipid plus RA/L surfactant molecules. Most of the POPC/C12En mixtures studied form a single lamellar liquid crystalline LR phase, which is preferred by the pure lipid component. However, high concentration of surfactant molecules with short EO chains (47) Ullrich, A. S.; Watts, A. Biophys. J. 1994, 66, 1441-1449.

Ko¨ nig et al.

can bring about HII phase formation, most likely by transfer of at least some of the surfactant molecules into the hydrocarbon region of the aggregates. The solubilization potential of C12En for the partially hydrated POPC studied here is reduced compared to the case of fully hydrated lipid. Glossary POPC C12En EO 2H NMR ∆νQ 31P

NMR

∆σ RH RW/L RA/L d dhc WL+A AL+A

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine oligo(ethylene oxide) mono-n-dodecyl ether ethylene oxide deuterium nuclear magnetic resonance spectroscopy absolute value of the deuterium quadrupolar splitting of 2H2O phosphorus nuclear magnetic resonance spectroscopy phosphorus chemical shift anisotropy relative humidity water/lipid molar ratio surfactant/lipid molar ratio lamellar repeat spacing membrane hydrocarbon core thickness water uptake per lipid plus RA/L surfactant molecules area requirement per lipid plus RA/L surfactant molecules

Acknowledgment. This work was supported by grants from the Deutsche Forschungsgemeinschaft in the framework of SFB 294 and from the Bundesministerium fu¨r Forschung und Technologie. The authors thank Professor Paltauf, Technical University Graz/Austria for synthesizing POPC. LA960571Y